There has been a protein in which an amino acid residue at a desired position is substituted with a non-natural amino acid and/or an α-hydroxy acid, and such protein is called a non-natural protein (or super protein). The non-natural protein has a new biological activity, catalytic activity, structure and function, which are not found in natural proteins. Because of these features, non-natural protein synthesis is an indispensable basic technology for overcoming limitations of conventional protein engineering.
In recent years, it has become possible to synthesize a non-natural protein by using Escherichia coli, yeast, insect cells, and mammalian cells. Usually, a non-natural amino acid is introduced at an amber codon (UAG codon) (which is one of stop codons), because it is not desirable that a non-natural amino acid be introduced at a non-specific position in such a synthesizing system.
It is known that the introduction of a non-natural amino acid at an amber codon competes with release factor, which terminates translation at a UAG codon. Non-Patent Literatures 1 and 2 disclose that weakening RF-1 activity (which is a release factor for Escherichia coli) improves introduction efficiency for a non-natural amino acid. Furthermore, Non-Patent Literature 3 discloses that decreasing the amount of RF-1 in cell-free protein synthesis system enables efficient introduction of a non-natural amino acid at an amber codon.
Furthermore, it is considered that introduction efficiency can be further improved by causing a prfA gene coding for RF-1 to be defective. As one method for causing the prfA gene to be defective, Non-Patent Literature 4 discloses a method including replacing amber codons of all genes on a genome with ocher codons or opal codons. Non-Patent Literatures 5 and 6 report Escherichia coli in which a prfA gene is caused to be defective by replacing all amber codons with ocher codons.